A viable, transparent corneal model can provide the basis for studying novel ophthalmic drugs and new gene delivery approaches. Moreover, there is currently a clinical need for improved understanding of corneal wound-healing mechanisms in order to solve corneal haze problems associated with LASIK procedures and outcomes in corneal transplants. An understanding of the factors that contribute to the expression of the wound-healing phenotype in the cornea can lead to an understanding of how to control these changes. The overall goal of this proposed project is to understand and control the relationship between corneal cell behavior and resulting transparency in a tissue-engineered corneal model. A number of input signals to the cells have been characterized, including chemical, mechanical, electromagnetic, and topographical signals. The results of these studies taken together indicate that physical signals are more promising than chemical signals for the control of the wound-healing response in corneal fibroblasts. The proposed studies aim at understanding the differential, and perhaps more importantly, the synergistic effects of several signals in order to determine which signals or signal combinations are most successful as we move toward our ultimate goal of a three-dimensional corneal model. Cells will be analyzed for protein levels by western blot, mRNA levels will be analyzed by quantitative real-time PCR (qR-T PCR), and the extent of light scattering will be determined by optical coherence microscopy (OCM). Specifically, we will assess signaling associated with the wound healing response, integrin signaling, alpha-SMA, TKT, ALDH1, and matrix remodeling markers associated with changes in the keratocyte phenotype under the various treatment conditions detailed in this proposal. Cell behavior will be assessed to understand (1) the relative importance of matrix alignment and matrix composition; (2) the effect of matrix stiffness; and (3) the potentially synergistic effectsof light and mechanical strain input signals. By understanding the relative strengths of these input signals, we can more intelligently construct a transparent corneal matrix and model. Overall, these studies will lead to a better understanding of the role of biomimicry in the design and implementation of tissue-engineered systems. In addition, they will contribute to the understanding of wound healing in fibroblastic cells.